L-2-hydroxyglutaric aciduria: identification of ten novel mutations in the L2HGDH gene.

L-2-hydroxyglutaric aciduria (L-2-HGA) is a metabolic disease with an autosomal recessive mode of inheritance. It was first reported in 1980. Patients with this disease have mutations in both alleles of the L2HDGH gene. The clinical presentation of individuals with L-2-HGA is somewhat variable, but affected individuals typically suffer from progressive neurodegeneration. Analysis of urinary organic acids reveals an increased signal of 2-hydroxyglutaric acid, mainly as the L-enantiomer. L-2-HGA is known to occur in individuals of various ethnic backgrounds, but up to now mutation analysis has been mainly focused on patients of Turkish and Portuguese origin. This led us to confirm the diagnosis on the DNA level and undertake the corresponding mutation analysis in individuals of diverse ethnicity previously diagnosed with L-2-HGA on the basis of urinary metabolites and clinical/neuroimaging data. In 24 individuals from 17 families with diverse ethnic and geographic origins, 13 different mutations were found, 10 of which have not been reported previously. At least eight of the patients were compound heterozygotes. The identification of two mutations (c.751C > T and c.905C > T in exon 7) in patients with different origins supports the view that they occurred independently in different families. In contrast, the mutation c.788C > T was detected in all six Venezuelan patients originating from the same Caribbean island of Margarita, but not in other patients, thus rendering a founder effect likely. None of the mutations was found in the control population, indicating that they are most probably causative. Mutation analysis may improve the quality of diagnosis and prenatal diagnosis of L-2-HGA.

Guanidinoacetate administration increases acetylcholinesterase activity in striatum of rats and impairs retention of an inhibitory avoidance task.

Guanidinoacetate methyltransferase deficiency (GAMT-deficiency) is an inborn error of metabolism biochemically characterized by accumulation of guanidinoacetate (GAA) and depletion of creatine; the pathogenesis of brain dysfunction in this disorder is not yet established. In the present study we investigated the effect of intrastriatal administration of GAA on acetylcholinesterase (AChE) activity and on memory acquisition, consolidation and retrieval of step-down inhibitory avoidance task in rat. Results showed that GAA significantly increased AChE activity in rat striatum 30 min (50%) and 3 h (25%), but not 6 h after drug administration. GAA impaired test session performance when applied 30 min before training or after training, and before testing sessions, i.e., impaired memory acquisition, consolidation and retrieval. When injected with a 6 hour interval, GAA affected only memory retrieval. Although the mechanisms of action of GAA on AChE activity and on memory are unclear, these findings suggest that the accumulation of GAA found in patients with GAMT-deficiency may be one of the mechanisms involved in neural dysfunction. Further studies are necessary to evaluate these mechanisms.

Evidence that 3-hydroxyisobutyric acid inhibits key enzymes of energy metabolism in cerebral cortex of young rats.

3-Hydroxyisobutyric aciduria is an inherited metabolic disease caused by 3-hydroxyisobutyryl-CoA dehydrogenase deficiency. Tissue accumulation and high urinary excretion of 3-hydroxyisobutyric acid is the biochemical hallmark of this disorder. Clinical phenotype is heterogeneous and generally includes dysmorphic features, delayed motor development, profound mental impairment, and acute encephalopathy. Lactic acidemia is also found in the affected patients, indicating that mitochondrial dysfunction may be involved in the pathophysiology of this disorder. Therefore, the aim of the present work was to investigate the in vitro effect of 3-hydroxyisobutyric acid (0.1, 0.5 and 1mM) on essential enzymes of energy metabolism, namely the activities of the respiratory chain complexes I-V, total, cytosolic and mitochondrial creatine kinase and Na(+), K(+)-ATPase in cerebral cortex homogenates of 30-day-old rats. We also measured the rate of oxygen consumption in brain mitochondrial preparations in the presence of 3-hydroxyisobutyric acid. 3-Hydroxyisobutyric acid significantly reduced complex I-III (20%), without affecting the other activities of the electron transport chain. Furthermore, 3-hydroxyisobutyric acid did not change state III, state IV and the respiratory control ratio in the presence of glutamate/malate or succinate, suggesting that its effect on cellular respiration was weak. On the other hand, the activities of total and mitochondrial creatine kinase, but not cytosolic creatine kinase, were inhibited (30%) by 3-hydroxyisobutyric acid. We also observed that 3-hydroxyisobutyric acid-induced inhibition of mitochondrial creatine kinase activity was fully prevented by pre-incubation of the homogenates with reduced glutathione, alpha-tocopherol or the combination of superoxide dismutase plus catalase, suggesting that this inhibition was mediated by oxidation of essential thiol groups of the enzyme probably by superoxide, hydrogen peroxide and/or peroxyl radicals. It was also demonstrated that Na(+), K(+)-ATPase activity from synaptic plasma membranes was markedly suppressed (37%) by 3-hydroxyisobutyric acid and that this effect was prevented by alpha-tocopherol co-incubation implying that peroxyl radicals were probably involved in this action. Considering the importance of the affected enzyme activities for brain metabolism homeostasis and neurotransmision, it is suggested that increased tissue levels of 3-hydroxyisobutyric acid may contribute to the neurodegeneration of patients affected by 3-hydroxyisobutyric aciduria and possibly explain previous reports describing elevated production and excretion of lactate.

Evidence for oxidative stress in tissues derived from succinate semialdehyde dehydrogenase-deficient mice.

Animal models of inborn errors of metabolism are useful for investigating the pathogenesis associated with the corresponding human disease. Since the mechanisms involved in the pathophysiology of succinate semialdehyde dehydrogenase (SSADH) deficiency (Aldh5a1; OMIM 271980) are still not established, in the present study we evaluated the tissue antioxidant defences and lipid peroxidation in various cerebral structures (cortex, cerebellum, thalamus and hippocampus) and in the liver of SSADH-deficient mice. The parameters analysed were total radical-trapping antioxidant potential (TRAP) and glutathione (GSH) levels, the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), as well as thiobarbituric acid-reactive substances (TBARS). We first observed that the tissue nonenzymatic antioxidant defences were significantly reduced in the SSADH-deficient animals, particularly in the liver (decreased TRAP and GSH) and in the cerebral cortex (decreased GSH), as compared to the wild-type mice. Furthermore, SOD activity was significantly increased in the liver and cerebellum, whereas the activity of CAT was significantly higher in the thalamus. In contrast, GPx activity was significantly diminished in the hippocampus. Finally, we observed that lipid peroxidation (TBARS levels) was markedly increased in the liver and cerebral cortex, reflecting a high lipid oxidative damage in these tissues. Our data showing an imbalance between tissue antioxidant defences and oxidative attack strongly indicate that oxidative stress is involved in the pathophysiology of SSADH deficiency in mice, and likely the corresponding human disorder.

Tryptophan reduces creatine kinase activity in the brain cortex of rats.

Hypertryptophanemia is a rare inherited metabolic disorder probably caused by a blockage in the conversion of tryptophan to kynurenine, resulting in the accumulation of tryptophan and some of its metabolites in plasma and tissues of affected patients. The patients present mild-to-moderate mental retardation with exaggerated affective responses, periodic mood swings, and apparent hypersexual behavior. Creatine kinase plays a key role in energy metabolism of tissues with intermittently high and fluctuating energy requirements, such as nervous tissue. The main objective of the present study was to investigate the effect of acute administration of tryptophan on creatine kinase activity in brain cortex of Wistar rats. We also studied the in vitro effect of this amino acid on creatine kinase activity in the brain cortex of non-treated rats. The results indicated that tryptophan inhibits creatine kinase in vitro and in vivo. We also observed that the in vitro inhibition was fully prevented but not reversed by pre-incubation with reduced glutathione, suggesting that the inhibitory effect of tryptophan on CK activity is possibly mediated by oxidation of essential thiol groups of the enzyme and/or long-lasting adduct formation. Considering the importance of creatine kinase for the maintenance of energy homeostasis in the brain, it is conceivable that an inhibition of this enzyme activity in the brain may be one of the mechanisms by which tryptophan might be neurotoxic.